Analyses of the human proteome hold the promise of the ability for early identification of disease and for insights into the pathological processes involved (Berhane et al., 2005; Zhao et al., 2005; Veenstra et al., 2005). A difficult problem associated with this goal is the need to sample the proteins involved easily and to do so over a wide dynamic range (Anderson & Hunter, 2005; Vidal et al., 2005). For screening large populations, urine and plasma provide a convenient access to fluids equilibrated with total body metabolism. However, each of these sources presents unique problem sets in sample preparation.
Plasma contains high concentrations of both albumin and IgGs so that the detection and identification of non-(Alb+IgG) proteins is made difficult if the highly predominant species are not selectively removed. Affinity methods for their reduction in intact or diluted plasma have been reported (Echan et al., 2005; Greenough et al., 2004), but this removal may be accompanied by loss of peptides or other metabolites complexed to the highly abundant species (Zhou et al., 2004). Despite this problem, plasma offers a rich and relatively constant concentration of proteins in a milieu suitable for various analytical methods used in proteomic analysis.
Urine also provides a simple access to body fluids, but the analytical difficulties are quite different than plasma (Haubitz et al., 2005). Urine has temporal variations in urine protein, peptide, and metabolomic content that must be overcome by sampling pooled collections (Lenz et al., 2005). Excluding breach of the glomerular filtration unit, protein concentrations are generally much lower than in plasma and can be accompanied by highly variable electrolyte levels so that simple concentration without electrolyte separation is not indicated. More recently, the presence of hydrophobic, membrane proteins (Thongboonkerd et al., 2002), whose source has been traced to urinary exosomes (Pisitkun et al., 2004), has opened the possibility of preliminary separation of these particles from high molecular weight, but soluble, proteins and both of these from low molecular weight proteins and peptides.
Thus, there is an unmet need in the art for improved methods of isolating polypeptides from biological fluids for proteomic analysis. Further, there is an unmet need for improved methods of isolating exosomes from biological fluids, wherein the exosomes comprise a useful subset of biomarker polypeptides having applications in proteomic analysis generally and disease diagnosis and progression in particular.